Members of the Tumor Necrosis Factor (TNF) superfamily, including the Death Receptors and TNF Receptors, are actively being pursued as targets for cancer therapy, among other diseases. Recent cellular studies highlight the importance of a single methionine residue (Met 152) within Death Receptor 5, whereby mutation to alanine completely disrupts ligand binding. While there is yet no explanation for this disruption, our interrogation of the crystal structure has revealed that the sulfur atom in DR5-Met152 is in close proximity (~ 5 A) to a tyrosine (Tyr237) in the TRAIL ligand. Additionally, Surface Plasmon Resonance measurements have shown that mutation of TRAIL-Tyr237 to alanine causes a five-fold increase in the ligand-receptor dissociation constant. A search of the protein data bank suggested an optimal geometry for the interaction between sulfur and aromatic groups in proteins, and subsequent quantum chemistry calculations have suggested significant stabilization due this interaction motif. Despite this, there has been little (or no) attention paid to this potentially important motif in the cell biology literature. Based upon available crystal structure data, we have recently discovered that a similar sulfur-aromatic interaction exists in another of the TNF ligand-receptor pairs, namely lymphotoxin-alpha (LT1) bound to TNF-R1. Other than our observations and preliminary results, no information yet exists regarding this interaction in TNF-R1, nor has any work been geared toward understanding the functional significance or the chemical foundations of the sulfur-aromatic motif in TNF receptors in general. It is our hypothesis that interactions between the sulfur atom in methionine and aromatic residues underlie a highly stabilizing and targetable binding motif within the ligand-receptor pairs of the TNF superfamily. This grant proposes to complement our computational findings with experimental molecular and cellular biology studies in order to validate the biological (and potential pharmaceutical) importance of the sulfur-aromatic interaction. PUBLIC HEALTH RELEVANCE: Tumor Necrosis Factor Superfamily proteins are involved in a wide range of diseases, including cancers and auto-immune diseases. This work will reveal new and critical chemical details about these proteins that can then be used for designing new treatments.